TLR3 and TLR7 Are Targeted to the Same Intracellular Compartments by Distinct Regulatory Elements*♦

Toll-like receptor (TLR) 3 and TLR7 are indispensable for host defense against viral infection by recognizing virus-derived RNAs and are localized to intracellular membranes via an unknown mechanism. We recently reported experiments with chimeric Toll-like receptors that suggested that the subcellular distribution of TLRs may be defined by their transmembrane and/or cytoplasmic domains. Here we demonstrate that the intracellular localization of TLR3 is achieved by a 23-amino acid sequence (Glu727 to Asp749) present in the linker region between the transmembrane domain and Toll-interleukin 1 receptor resistance (TIR) domain. In contrast, the intracellular localization of TLR7 is achieved by its transmembrane domain. These elements also targeted a heterologous type I transmembrane protein CD25 to the intracellular compartment that contained TLR3 and TLR7. Despite their using distinct regulatory elements for intracellular localization, TLR3 was found to co-localize with TLR7. In addition, TLR3 and TLR7 were preferentially localized near phagosomes containing apoptotic cell particles. These findings reveal that TLR3 and TLR7 contain unique targeting sequences, which differentially lead them to the same intracellular compartments and adjacent to phagosomes containing apoptotic cell particles, where these receptors may access their ligands for the induction of immune responses against viral infection.

Toll-like receptors (TLRs) 2 are pattern-recognition receptors that detect highly conserved molecular structures of microorganisms or viruses and regulate both innate and adaptive immune responses against pathogens (1). More than 10 TLRs have been identified in human and mouse (2). TLR1, TLR2, TLR4, TLR5, and TLR6 recognize bacterial cell wall and cell surface components, such as lipoproteins, lipopolysaccharide, and flagellin. On the other hand, TLR3, TLR7, TLR8, and TLR9 recognize pathogen nucleic acids, such as viral RNAs and bacterial DNA (2,3). All TLRs have a cytoplasmic signaling domain called the Toll/interleukin 1 receptor resistance (TIR) domain, which associates with intracellular TIR domain-containing adaptors, such as MyD88, TIRAP, TRIF/TICAM1, and TRAM/TICAM2. These TLR-associated adaptor molecules in turn mediate downstream signaling to induce pro-inflammatory and/or anti-viral innate immune responses (3).
Since all TLRs are typical type I transmembrane proteins composed of an NH 2 -terminal signal peptide, an extracellular domain involved in ligand recognition, a single transmembrane domain, and a cytoplasmic domain, it was initially assumed that all TLRs would be expressed on the cell surface. However, studies using chimeric receptor approaches, fluorescently labeled TLRs, and anti-TLR antibodies have indicated that whereas TLR1, TLR2, TLR4, TLR5, and TLR6 are expressed on the cell surface, TLR3, TLR7, and TLR9 are completely localized in intracellular acidic compartments (4 -9). Based on data with chimeric receptors, TLR8 appears to be localized primarily intracellularly but with a small fraction on the cell surface (4). How intracellular targeting for the intracellular TLRs is achieved is unknown; the typical sequences for targeting proteins to intracellular compartments, such as H/KEDL for targeting to the endoplasmic reticulum (ER), have not been identified in the intracellular TLRs.
Upon viral infection, phagocytes such as macrophages and dendritic cells take up virus-infected apoptotic cells. Viral RNAs may be released from these apoptotic cells in intracellular acidic compartments, such as phagosomes, by the action of lysosomal enzymes. Accordingly, it is attractive to propose that TLR3 and TLR7, which are receptors for viral RNAs and are indispensable for mounting anti-viral innate immune responses by inducing type I interferons (10,11), are localized to phagosomes or are delivered there after uptake of particles.
Here, we utilized TLR chimeric receptors composed of the extracellular region of TLR4 fused to the transmembrane and cytoplasmic regions of TLR3 or TLR7 to identify the regions of these proteins crucial for their intracellular localization. Those chimeras were readily detected with an anti-mouse TLR4/MD-2 monoclonal antibody and co-localized with wild-type TLRs. We found that the intracellular localization of TLR3 and TLR7 was achieved by distinct elements: the linker region for TLR3 and the transmembrane region for TLR7. These elements also targeted a heterologous type I transmembrane protein CD25 to the same intracellular compartment as TLR3 and TLR7. Although they used different regulatory elements, TLR3 and TLR7 were localized in the same intracellular compartments and were preferentially localized close to phagosomes containing apoptotic particles. These studies not only map the molecular determinants of the intracellular localization of TLR3 and TLR7 but also provide evidence of unique targeting sequences of type I transmembrane proteins for subcellular distribution.

EXPERIMENTAL PROCEDURES
Mice and Cell Culture-C57BL/10ScN mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and bred in our colony. Bone marrow-derived macrophages (BMDMs) were prepared and maintained as described previously (4). BMDMs at day 4 or 5 after isolation from the bone marrow were used for retroviral infection. The infection efficiency was generally 35-40%. HEK293T cells and mouse 3T3 fibroblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.
DNA Constructs and Stable Cell Lines-The murine TLR4/TLR chimeras were described previously (4). Truncated forms of TLR4/TLR3 and TLR4/TLR7 chimeras were amplified by PCR and then subcloned into the pMXpie bicistronic retroviral vector, which encodes the gene of interest followed by an internal ribosomal entry site element and the enhanced green fluorescent protein (GFP) gene (12). GFP fluorescence is therefore an indication of infection efficiency. The transmembrane domain swap mutants of TLR4/TLR chimeras were prepared by multiple PCRs using overlapping sequences and then subcloned into pMXpie. Murine CD25 cDNA was amplified by PCR from mouse spleen cDNA. cDNAs encoding CD25-TLR3 linker fusions and CD25-TLR7 chimeras were prepared by multiple PCRs using overlapping sequences and then subcloned into pMXpie. The COOH-terminal yellow fluorescent protein (YFP)-or cyan fluorescent protein (CFP)-tagged TLR3 (TLR3 YFP ), TLR4 (TLR4 YFP ), TLR7 (TLR7 CFP and TLR7 YFP ), TLR4/ TLR3 chimera (TLR4/TLR3 CFP ), CD25 (CD25 CFP ), CD25-EG-ID fusion (EG-ID CFP ), or CD25-TLR7 chimera 1 (chimera 1 CFP ) were constructed by fusing cDNA encoding the full-length TLR3, TLR4, TLR7, TLR4/ TLR3 chimera, CD25, CD25-EG-ID, or chimera 1 to cDNA encoding YFP or CFP via a short linker sequence ((Gly-Gly-Gly-Gly-Ser)ϫ3). They were cloned into the pMXrmv5 retroviral vector, which was prepared by deleting the internal ribosomal entry site-enhanced GFP sequence from pMXpie. Murine MD-2 cDNA was kindly provided by Dr. K. Miyake (University of Tokyo). Murine CD14 cDNA was amplified by PCR from mouse liver cDNA. cDNAs encoding CD14 and MD-2 were cloned into the pMXrmv5I bicistronic retroviral vector, which was created by inserting an internal ribosomal entry site sequence into pMXrmv5.
HEK293T cells or 3T3 fibroblasts expressing both murine CD14 and MD-2 were prepared by retroviral gene transfer as described below. Both CD14-and MD-2-positive cells were selected for growth in medium containing 2-4 g/ml puromycin for a week, and individual clones were tested by introducing TLR4 transiently and staining with phycoerythrin (PE)-conjugated anti-mouse CD14 antibody (Pharmingen) or anti-mouse TLR4/MD-2 antibody (clone MTS510, eBioscience, San Diego, CA). HEK293T cells and 3T3 fibroblasts expressing both murine CD14 and MD-2 are referred to as 293TCM and 3T3CM, respectively.
Viral Production and Infection-Introduction of all genes indicated in this study into various cell types was carried out by retroviral gene transfer as described previously (4), unless otherwise stated. Briefly, retroviruses were produced by triple transfection of HEK293T cells with retroviral constructs along with gag-pol and vesicular stomatitis virus G glycoprotein expression constructs (13). Viral supernatants were collected 48 h after transfection and added to the cells. Cells and viruses were then centrifuged at 1,800 rpm for 45 min (293T and 3T3 cells) or 2,400 rpm for 1 h (BMDMs) at room temperature followed by incubation for 6 h at 37°C in 5% CO 2 -95% air. Then, viral supernatants were replaced with fresh culture media.
Random Mutagenesis and Isolation of Mutant Clones with Altered Localization-Random mutagenesis of the transmembrane and cytoplasmic domains of TLR4/TLR3 and TLR4/TLR7 chimeras was carried out as described in the legend to Fig. 2A with the Genemorph II Random Mutagenesis Kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. Primers used for mutagenesis of both TLR3 and TLR7 sequences were 5Ј-ACGCGTCGACTTTAATAATTCTACCTGT-3Ј (forward) and 5Ј-AATTTACGTAGCGGCCGCGGCGCGCCGGCC-CTCGAG-3Ј (reverse). PCR products were digested with SalI and NotI and then ligated into the XhoI/NotI site of pMXpie-TLR4(EX), which contained the cDNA encoding amino acids 1-598 of TLR4. OmniMAX supercompetent cells (Invitrogen) were transformed with the ligation mixture and plated onto 5 ϫ 15 cm LB-ampicillin agar plates. About 40,000 bacterial colonies were collected and expanded in LB media, and the plasmid DNAs were purified from the pooled bacteria. This DNA was used to generate a pool of retroviruses encoding mutated TLR-4/TLR chimeras (TLR4/TLR Mu ). 293TCM cells were infected with viruses, and the cells expressing TLR4/TLR Mu chimeras on the cell surface were enriched by cell sorting by using a MoFlow cell sorter (Dako Cytomation, Carpinteria, CA) using PE-conjugated anti-mouse TLR4/-MD-2 antibody. Cells obtained from the second sort were then cloned by limiting dilution. Single clones were isolated, and the cell surface level of TLR4/TLR Mu chimera in each clone was determined by flow cytometric analysis using anti-mouse TLR4/MD-2 antibody. Total RNA was prepared from GFPϩTLR4/MD-2ϩ clones and was reverse-transcribed to prepare cDNAs as described previously (4). cDNAs encoding the transmembrane and cytoplasmic domains of TLR4/TLR Mu chimeras were amplified by PCR, and the mutation in each clone was determined by DNA sequencing.
Microscopy-BMDMs expressing YFP or CFP fusion proteins were split onto glass coverslips. The next day, the cells were treated with Alexa Fluor 594-conjugated cholera toxin subunit b (CTXb; Molecular Probes, Eugene, OR) for 20 min on ice. After washes, the images were acquired using a Deltavision deconvolution microscope (Applied Precision, Issaquah, WA) or a Carl Zeiss LSM510 META laser-scanning confocal microscope. The computational deconvolution was carried out with the SoftWoRX software.
To test the co-localization of phagocytic particles with TLRs in macrophages, apoptotic T cells were prepared and added to BMDMs. Briefly, cells were prepared from the whole spleen derived from C57BL/ 10ScN mice and were stimulated with 10 g/ml anti-CD3 antibody (Pharmingen). Three days later, cells were washed with media and were cultured another 3 days without anti-CD3 antibody. The apoptotic T cells were stained with 7-AAD (Pharmingen) and then added to BMDMs expressing TLR3 YFP or TLR7 YFP at 5:1 ratio of T cells to macrophages. After incubation for 30 min, the BMDMs were washed with PBS containing 2% fetal calf serum and 0.09% sodium azide 5 times, and the images were acquired as described above.
Flow Cytometric Analysis-The cell surface distribution of TLR4/ TLR chimeras and CD25-TLR fusion or chimeric proteins was assessed by flow cytometric analysis using PE-anti-mouse TLR4/MD-2 antibody and PE-anti-mouse CD25 antibody (BD Pharmingen), respectively. To assess intracellular expression levels, cells were fixed and permeabilized and then stained with fluorescent antibodies, as described previously (4).

RESULTS
TLR3 and TLR7 Are Targeted to an Intracellular Compartment by Their Transmembrane and Cytoplasmic Domains-Recent data have suggested that TLR3 and TLR7 are localized in intracellular acidic compartments, such as phagosomes, and recognize viral nucleic acids, such as double-and single-stranded RNA derived from virus-infected apoptotic cells or virus particles (4,6,7,11,14,15). Consistent with these reports, we found that TLR3 and TLR7 molecules containing a YFP moiety fused to their COOH termini were localized intracellularly but not on the cell surface (Fig. 1A). In contrast, a fusion of TLR4 with YFP (TLR4 YFP ) was expressed both in internal membranes and on the cell surface, which was visualized by staining ganglioside GM1 with fluores-cent CTXb (8) (Fig. 1A). The TLR4 YFP , TLR3 YFP , and TLR7 YFP molecules were functional, as they mediated normal responses to lipopolysaccharide, poly(I:C), and loxoribine, respectively, in 293TCM cells (data not shown).
We had previously observed that chimeric receptors, which are composed of the extracellular region of TLR4 fused to the transmembrane and cytoplasmic regions of TLR3 or TLR7, were not detected on the cell surface of BMDMs ( Fig. 1B and Ref. 4). To test whether the TLR4/TLR chimeras behave like full-length TLRs, both TLR3 YFP and TLR4/ TLR3 CFP were expressed in the same BMDMs, following staining with CTXb, and their subcellular distribution was determined microscopically. As shown in Fig. 1C, the TLR4/TLR3 chimera was clearly colocalized with TLR3 in cytoplasmic vesicles but not co-localized with CTXb at the cell surface, indicating that the TLR4/TLR chimeras may properly reflect the localization of TLRs and therefore that the transmembrane and/or cytoplasmic domains of TLR3 are responsible for this intracellular localization.
Random Mutagenesis of the Transmembrane and Cytoplasmic Domains of TLR4/TLR Chimeras-We hypothesized that TLR3 and TLR7 are targeted to intracellular compartments by sequences present in their transmembrane and/or cytoplasmic domains. This hypothesis predicts that mutation of the targeting sequence would allow the intracellular TLR to come to the cell surface. Since the subcellular distribution of the TLR4/TLR chimeras was easily determined by flow cytometric analysis using anti-mouse TLR4/MD-2 antibody, we decided to use these chimeric receptors to investigate the putative targeting signals. cDNAs encoding the transmembrane and cytoplasmic domains of the TLR4/TLR chimeras were randomly mutated by error-prone PCR and put back into the context of the TLR4 chimera ( Fig. 2A). Random sequencing of plasmids encoding the mutated TLR chimeras confirmed that the mutations were randomly generated throughout the transmembrane and cytoplasmic domains of the TLR4/TLR chimeras (data not shown). The mutation rates of the TLR4/TLR3 and TLR4/TLR7 chimeras were 1.7 Ϯ 0.3 and 2.9 Ϯ 0.4 per clone at the nucleotide level and 1.1 Ϯ 0.4 and 1.6 Ϯ 0.3 per clone at the amino acid level, respectively. The pool of mutated chimeras was introduced into HEK293T cells stably expressing mouse CD14 and MD-2 (293TCM cells), because 1) transfected mouse TLR4 was detected on the cell surface in greater amounts in 293TCM cells than in 293T cells (data not shown), 2) the TLR4/TLR3 and TLR4/TLR7 chimeras were detected intracellularly in 293TCM cells similarly to BMDMs (data not shown), and 3) the high susceptibility of these cells to retrovirus infection enabled us to screen a large number of TLR4/TLR mutant chimeras. Retroviral vectors producing different TLR4/TLR mutant chimeras were prepared from about 40,000 different transformed Escherichia coli colonies. The 293TCM cells were infected with retroviruses containing the mutated TLR4/TLR3 or TLR4/TLR7 chimeras, and the cell surface expression level of the chimeras was determined by flow cytometric analysis using FIGURE 1. TLR3 and TLR7 are targeted to intracellular compartments by their transmembrane and/or cytoplasmic domains. A, TLR3 and TLR7, but not TLR4, are retained intracellularly in BMDMs. The BMDMs expressing TLR3 YFP , TLR4 YFP , or TLR7 YFP were stained with Alexa Fluor 594-conjugated CTXb. Images were acquired using a Deltavision deconvolution microscope. TLR3, TLR4, and TLR7 are shown in green (pseudo-color), and CTXb is shown in red. Regions with co-localization appear yellow. Higher magnification panels to the right show the red and green channels separately for the boxed area. B, both TLR4/TLR3 and TLR4/TLR7 chimeras are not detected on the cell surface. The expression of TLR chimeras in BMDMs was determined by flow cytometric analysis using anti-mouse TLR4/MD-2 antibody as described under "Experimental Procedures." The GFP-positive cells were gated and displayed for the TLR chimera staining. Intact, cells without fixation/permeabilization; Fixed/Perm, cells with fixation/permeabilization. Gray, isotype control; line, anti-TLR4/MD-2 antibody. C, the TLR4/TLR3 chimera co-localizes with TLR3. BMDMs expressing both TLR3 YFP and TLR4/TLR3 CFP were stained with Alexa Fluor 594-CTXb, and images were acquired as described for A. TLR3, TLR4/ TLR3 chimera, and CTXb are shown in green, red (pseudocolor), and blue (pseudocolor), respectively. The boxed area is enlarged in the right panels, which show green, red, and merged versions of the image. Merged images with red and green co-localization result in yellow color. NOVEMBER 4, 2005 • VOLUME 280 • NUMBER 44 FIGURE 2. Random mutagenesis of the transmembrane and cytoplasmic domains of TLR4/TLR chimeras. A, random mutagenesis of the transmembrane and cytoplasmic domains of TLR4/TLR3 and TLR4/TLR7 chimeras by error-prone PCR. The transmembrane and cytoplasmic domains of TLR3 or TLR7 were amplified by error-prone PCR using primers indicated by the arrows, and then the PCR products were cloned into the pMXpie-TLR4(EX) vector as described under "Experimental Procedures." The mutation rates of the TLR4/TLR3 and TLR4/TLR7 chimeras were 1.7 Ϯ 0.3 and 2.9 Ϯ 0.4 per clone at the nucleotide level and 1.1 Ϯ 0.4 and 1.6 Ϯ 0.3 per clone at the amino acid level, respectively. The stars show random anti-mouse TLR4/MD-2 antibody. Interestingly, a small population of GFPϩTLR4/MD-2ϩ cells was detected in 293TCM cells infected with the TLR4/TLR3 mutant chimera-producing retroviruses, but not wildtype chimera-producing retroviruses (Fig. 2B), suggesting that some TLR3 mutations had interfered with the normal intracellular targeting. In contrast, a GFPϩTLR4/MD-2ϩ population was not detected in 293TCM cells expressing TLR4/TLR7 mutant chimeras, although we tried to obtain a positive population by two rounds of cell sorting ( Fig.  2C and data not shown).

Distinct Intracellular Targeting Elements for TLR3 and TLR7
Region of the Cytoplasmic Domain Responsible for Intracellular Localization of TLR3-To isolate the cells expressing mutated TLR4/ TLR3 molecules that were expressed on the cell surface, the GFPϩTLR4/MD-2ϩ population was enriched by two rounds of cell sorting, and then about 200 single clones were isolated by limiting dilution. Sixty-five clones were confirmed to express TLR4/TLR3 chimera on the cell surface by flow cytometric analysis, and the mutation in each clone was determined by DNA sequencing (Fig. 3A). Interestingly, all mutant chimeras contained either point mutations giving rise to premature stop codons or short deletions or insertions leading to frameshifts. These mutations had the result of deleting the whole TIR domain and a part of the linker region between the transmembrane segment and the TIR domain. No full-length chimeras with point mutations were obtained in this screen.
To confirm the results of random mutagenesis, several truncation mutants of the TLR4/TLR3 chimeric receptor were created by sitedirected mutagenesis, and their subcellular distributions were examined in BMDMs from TLR4-deficient mice. As shown in Fig. 3C, the chimeras lacking the region from Ile 748 to the COOH termini were detected on the cell surface. In contrast, the chimeras that are slightly longer than the Ile 748 3 STOP mutant were localized intracellularly, suggesting that a short region in the cytoplasmic domain prior to the TIR domain is sufficient for the intracellular localization of TLR3.
Intracellular Localization of the TLR4/TLR7 Chimera Lacking Its Entire Cytoplasmic Domain-In contrast to the TLR4/TLR3 chimera, a GFPϩTLR4/MD-2ϩ population was not detected in the pool of randomly mutated chimeras (Fig. 2C) even after two rounds of sorting for potentially positive cells (data not shown). To determine whether either the linker region and/or TIR domain is necessary for the intracellular localization of TLR4/TLR7 chimera, several truncated mutants of the TLR4/TLR7 chimera were created by site-directed mutagenesis as shown in Fig. 4A, and the subcellular distributions of these mutated chimeras in BMDMs were determined by flow cytometric analysis using anti-mouse TLR4/MD-2 antibody. All mutants were still localized intracellularly (Fig. 4B), suggesting that the transmembrane region of TLR7 was sufficient to cause the intracellular localization of the TLR4/ TLR7 chimera.
The Cytoplasmic Linker Region of TLR3 Is Sufficient to Mediate Intracellular Localization of a Heterologous Transmembrane Protein-To determine whether the cytoplasmic linker region of TLR3 might be sufficient for targeting this receptor to an intracellular compartment, the Glu 727 to Asp 749 sequence from TLR3 and various truncated versions of it were fused to the COOH terminus of CD25 (interleukin 2 receptor ␣ chain), which is a type I transmembrane receptor with a short cytoplasmic region (14 amino acids) that is normally expressed on the cell surface (16). The resulting fusion proteins were expressed in BMDMs, and their subcellular distributions were determined by flow cytometric analysis using anti-mouse CD25 antibody. The cell surface expression of CD25 was significantly attenuated by fusing it to the TLR3 linker region (CD25-EG-ID, containing Glu 727 to Asp 749 of TLR3). Expression of CD25 on the cell surface was reduced about 4-fold by the presence of the linker region of TLR3. Removal of additional residues from either end of the TLR3 linker region abolished this partial intracellular retention (Fig. 5, B and C). Similar results with the CD25-TLR3 fusion proteins were seen in 293TCM cells and 3T3CM cells (data not shown).
We further examined whether the linker region of TLR3 targets CD25 to an intracellular compartment where TLR3 is located. Both TLR3 YFP and CD25 CFP or CD25-EGϳID CFP were expressed in the same BMDMs, following staining with CTXb, and their subcellular distribution was determined microscopically. As shown in Fig. 5D, the CD25 CFP was clearly co-localized with CTXb, while CD25-EG-ID CFP was less co-localized with CTXb, but was strongly co-localized with TLR3 YFP in the cytoplasm. Taken together, these results suggest that a part of the linker region (Glu 727 to Asp 749 ) contains an important motif responsible for targeting not only TLR3 but also a heterologous type I transmembrane protein to an intracellular compartment.
The Transmembrane Domain of TLR7 Is Sufficient to Mediate Intracellular Localization of a Heterologous Transmembrane Protein-TLR4/TLR7 chimeras with truncation of the TLR7 cytoplasmic domain were still retained intracellularly in primary macrophages (Fig. 4). This result suggested that the transmembrane domain, but not the linker region or TIR domain, is involved in the intracellular localization of TLR7. To test whether the transmembrane domain of TLR7 is capable of targeting a heterologous transmembrane protein to an intracellular compartment, the transmembrane region of CD25 was replaced with that of TLR7 as shown in Fig. 6A, and the localization of the resulting chimeric CD25 molecules containing or lacking the CD25 cytoplasmic domain in BMDMs was determined. The cell surface expression of CD25 was nearly completely prevented when the transmembrane domain of TLR7 was introduced into CD25 (Fig. 6, B and C). Similar results were seen in 293TCM cells and 3T3CM cells (data not shown).
We further examined whether the transmembrane domain of TLR7 targets CD25 to an intracellular compartment where TLR7 is located. Both TLR7 YFP and CD25 CFP or chimera 1 CFP were expressed in the same BMDMs, following staining with CTXb, and their subcellular distributions were determined microscopically. As shown in Fig. 6D, chimera 1 CFP was strongly co-localized with TLR7 YFP in the cytoplasm, but not co-localized with CTXb on the cell surface, suggesting that the transmembrane domain, but not the linker region or TIR domain, is responsible for targeting not only TLR7 but also other type I transmembrane proteins to an intracellular compartment.
Region 2 of the Transmembrane Domain of TLR7 Is the Most Important Region for Intracellular Localization of TLR7-Since no mutants of TLR4/TLR7 chimera were detected on the cell surface in a random mutagenesis approach, in which the mutation rate was about 1.6 amino acid change per sequence (Fig. 2C), the intracellular localization of  TLR7 is probably not dependent on a single amino acid residue in its transmembrane domain. Therefore, we examined whether a subregion within the transmembrane domain is responsible for the intracellular localization of TLR7. The TLR4/TLR7 chimera in which most of the cytoplasmic region had been removed (TLR4/TLR7 TM ) was used, and subregions within the TLR7 transmembrane domain were replaced by corresponding regions of TLR3 (Fig. 7A). The resulting swap mutants were expressed in BMDMs, and their subcellular distributions were determined by flow cytometric analysis using anti-mouse TLR4/MD-2 antibody. Consistent with the data in Figs. 3 and 4, the TLR4/TLR3 TM chimera, which lacked a linker region and TIR domain, was detected on the cell surface, while the TLR4/TLR7 TM chimera was completely localized intracellularly (Fig. 7B). Interestingly, the replacement of Region 2 of TLR7 with the corresponding region of TLR3 restored the cell surface expression of TLR4/TLR7 TM chimera to about a 50% of that seen in the TLR4/TLR3 TM chimera (Fig. 7, B and C). The effect of Region 2 on intracellular localization was greater than that of the other regions tested. Similar results were seen in 293TCM cells (data not shown). These results suggest that Region 2 may be the most important for targeting TLR7 to an intracellular compartment, although other regions contribute to some degree.
TLR3 Co-localizes with TLR7 in Cytoplasmic Membranes-Since TLR3 and TLR7 appeared to be targeted to intracellular compartments by distinct regulatory elements, e.g. the linker region of the former and transmembrane region of the latter, we next examined whether TLR3 and TLR7 reside in the same intracellular compartments or different compartments. Both TLR3 YFP and TLR7 CFP were expressed in BMDMs, and their subcellular localization was examined by deconvolution microscopy. As shown in Fig. 8, TLR3 strongly co-localized with TLR7, suggesting that the distinct regulatory elements of these two proteins lead TLR3 and TLR7 to the same intracellular compartment.
Both TLR3 and TLR7 Were Preferentially Localized Near Phagosomes Containing Apoptotic Cell Particles-TLR3 and TLR7 have been shown to recognize viral nucleic acids, such as double-and single-stranded RNA, respectively (11,14,15). These nucleic acids may become available for recognition in phagocytes, such as macrophages and dendritic cells, when they take up virus-infected apoptotic cells and after initial digestion of the apoptotic cells by lysosomal hydrolases in intracellular acidic compartments, such as phagosomes. Therefore, we next examined whether TLR3 and TLR7 were recruited to phagosomes containing apoptotic cells. 7-AAD-labeled apoptotic T cells were added to BMDMs expressing TLR3 YFP or TLR7 YFP , and the localization of TLRs and apoptotic cell particles was examined microscopically. TLR3 was preferentially localized at the periphery of phagosomes containing apoptoic cellderived particles (Fig. 9A). The particles with weaker signal of 7-AAD and stronger signal of TLR3 YFP may have been taken up by macrophages earlier. Similarly to TLR3, TLR7 was localized near phagosomes containing cell particles in most cells (Fig. 9B, the left cell). These data suggest that intracellular compartments containing TLR3 and TLR7 may fuse to phagosomes.

DISCUSSION
All known mammalian TLRs have primary structures indicating that they are typical type I transmembrane proteins containing an NH 2terminal signal peptide, an extracellular region consisting mostly of leucine-rich repeats, a single transmembrane region, and a cytoplasmic region largely made up of the TIR signaling domain. Whereas some TLRs are expressed on the cell surface, surprisingly, data obtained with chimeric receptor approaches (4,9), fluorescently labeled TLRs (8), and some anti-TLR antibodies (6,8) have indicated that TLR3, TLR7, and TLR9 are localized intracellularly, and are not detected on the cell surface. Here we present studies aimed at characterizing the regions of TLR3 and TLR7 that mediate the intracellular localization of these innate immune receptors for viral nucleic acids. Our results reveal that distinct regulatory elements mediate the intracellular localization of these two TLRs: the cytoplasmic linker region for TLR3 and the transmembrane domain for TLR7. Interestingly, these distinctive localization signals target TLR3 and TLR7 to the same intracellular compartments and possibly also direct these TLRs adjacent to phagosomes containing apoptotic cells, where these TLRs may access their ligands.
Our previous results using a TLR chimeric receptor approach suggested that the transmembrane and/or cytoplasmic domain of TLRs may define both their subcellular distribution and their signaling properties (4). Although type I transmembrane proteins usually enter the secretory pathway in the ER, and are eventually translocated to the plasma membrane, some type I transmembrane proteins are retained in intracellular compartments, such as the ER or the Golgi apparatus (17). These intracellular type I transmembrane proteins typically possess intracellular retention signals such as the H/KEDL and di-lysine motifs, FIGURE 5. A 23-amino acid sequence (Glu 727 to Asp 749 ) in the linker region of TLR3 attenuates the cell surface expression of CD25. A, schematic illustration of constructs of various CD25-TLR3 linker fusion proteins. Various lengths of the TLR3 linker region were fused to the COOH terminus of CD25. B and C, BMDMs from C57BL/10ScN mice were infected with retroviruses producing fusion proteins, and the subcellular distributions of the fusion proteins were determined by flow cytometry using a PE-anti-mouse CD25 antibody. Gray, isotype control; line, anti-mouse CD25 antibody. The cell surface level of each fusion protein was quantified as a ratio of mean fluorescence intensity of "intact" versus "fixed and permeabilized (Perm.)". These values were calculated after subtraction of mean fluorescence intensity of isotype control from that of anti-CD25 antibody staining. WT, full-length CD25 without any TLR sequences. D, BMDMs expressing both TLR3 YFP and CD25 CFP or CD25-EG-ID CFP were stained with Alexa Fluor 594-CTXb, and images were acquired by a Carl Zeiss LSM510 META laser-scanning confocal microscope. TLR3 is shown in green, CD25 and CD25-EGϳID are shown in red (pseudocolor), and CTXb is shown in light blue (pseudocolor).
which are located at the COOH terminus of the protein (17). Our studies indicate that neither TLR3 nor TLR7 are targeted to intracellular membranes by any of these well characterized targeting motifs and moreover that these two molecules have distinct targeting motifs that nonetheless target these TLRs to the same intracellular compartment.
In the case of TLR3, we were able to narrow the targeting signal down to a 23-amino acid region from E 727 to D 749 in the linker region between the transmembrane domain and the TIR domain. This 23-amino acid region was able to confer a substantial degree of intracellular localization to the heterologous type I transmembrane protein CD25, with cell surface expression reduced by ϳ4-fold. Furthermore, this region targeted CD25 to an intracellular compartment where TLR3 was located. Shortening either end of this motif substantially abolished its function in intracellular retention of CD25, and, in the case of COOH-terminal truncations, in the retention of the TLR4/TLR3 chimera. Thus, this 23-amino acid sequence may be the minimum requirement for targeting TLR3 to an intracellular compartment. This 23-amino acid sequence contains residues recently identified as important for TLR3 intracellular localization by Funami et al. (5). They mutated in pairs Arg 740 -Ile 741 , Phe 745 -Lys 746 , or Glu 747 -Ile 748 in human TLR3 lacking the TIR domain (TLR3⌬TIR) to alanines and found that the resulting mutated TLR3 molecules were mislocalized to the cell surface in HEK293 cells and Ba/F3 cells. Surprisingly, they found that critical res-idues for intracellular localization of TLR3⌬TIR in 293T cells were different from those in Ba/F3 cells, suggesting the existence of different sorting mechanisms in those cells. We did not observe any differences in cell surface expression of TLR4/TLR3 mutant chimeras between 293T/ CD14/MD-2 cells, 3T3/CD14/MD-2 cells, and BMDMs, indicating that the same sequence is important in these different types of cells. Some transmembrane proteins have multiple different intracellular targeting motifs. For example, the intracellular localization of the cationdependent mannose 6-phosphate receptor (CD-M6PR) is controlled by three different motifs in its cytoplasmic domain (18 -20). CD-M6PR functions to transport newly synthesized acid hydrolases from the trans-Golgi network to an acidified endosomal compartment. CD-M6PR binds M6P-modified hydrolases in the trans-Golgi network and then moves with them to late endosomes, where it releases its M6Phydrolase cargo and returns to the Golgi to repeat the process or alternatively moves to the plasma membrane where it is rapidly internalized via clathrin-coated vesicles (21,22). A di-leucine motif (Leu 256 -Leu 257 ) near its carboxyl terminus is required for efficient entry into Golgi clathrin-coated pits (19). Two other signals, Phe 224 -X-X-X-X-Phe 229 and Tyr 256 -X-X-Val 259 , mediate the rapid internalization at the plasma membrane (18). We found that the 23-amino acids sequence (Glu 727 to Asp 749 ) in the linker region of TLR3 is sufficient to mediate intracellular retention both of the TLR4/TLR3 chimera and of the heterologous type FIGURE 6. Replacement of the transmembrane region of CD25 with that of TLR7 greatly reduces the cell surface expression of CD25. A, schematic illustration of constructs of CD25-TLR7 chimeric proteins. The portion coming from TLR7 corresponding to its transmembrane domain is indicated in gray. B and C, the subcellular distributions of the chimeric proteins were determined and quantified as described in Fig. 5C. D, BMDMs expressing both TLR7 YFP and CD25 CFP or chimera 1 CFP were stained with Alexa Fluor 594-CTXb, and images were acquired as described in the legend to Fig. 5D. TLR7 is shown in green, CD25 and chimera 1 are shown in red (pseudocolor), and CTXb is shown in light blue (pseudocolor).
I transmembrane protein CD25. Since a Y-X-X-V motif is present in this sequence, we tested whether TLR3, like CD-M6PR, is first localized on the plasma membrane and then immediately endocytosed in a clathrindependent manner. PE-anti-TLR4/MD-2 antibody was added to BMDMs expressing the TLR4/TLR3 chimera, the cells were incubated for 2 h at 37°C, and then the level of endocytosed TLR4/TLR3 chimera was assessed by flow cytometry. However, no endocytosis of the TLR4/ TLR3 chimera was detected by this method (data not shown), suggesting that TLR3 is not routed to the plasma membrane, even transiently. CD-M6PR has been shown to move between the trans-Golgi network and endosomes, while TLR3 selectively accumulated in multivesicular bodies-like vesicles (6), but was not co-localized with any particular markers for cytoplasmic organelles including endosomes (5). Thus, the mechanism for the intracellular localization of TLR3 appears to be different from that for the CD-M6PR. It should be noted, however, that although we have identified regions of TLR3 and TLR7 that are sufficient for intracellular targeting, we cannot at this time rule out the presence of additional targeting motifs in these molecules.
In contrast to the situation with TLR3, we found that TLR7 is local-ized to intracellular membranes by its transmembrane domain. Truncations of the TLR4/TLR7 chimera removing almost the entire intracellular domain were still retained intracellularly, and moreover, the TLR7 transmembrane domain was able to target another type I transmembrane protein CD25 to an intracellular compartment, where the targeted CD25 strongly co-localized with TLR7. It has been suggested that some type II transmembrane proteins, such as ␤-galactoside ␣2,6-  . TLR3 co-localizes with TLR7. BMDMs were infected with retroviruses producing TLR3 YFP and TLR7 CFP . 48 h after infection, fluorescence images were acquired using a Deltavision deconvolution microscope. TLR3 YFP is shown in green, and TLR7 CFP is shown in red (pseudocolor). Higher magnification panels to the right show the red, green, and merged channels separately for the boxed region. . Both TLR3 and TLR7 are preferentially localized adjacent to phagocytosed apoptotic T cells in BMDMs. A and B, apoptotic T cells were labeled with 7-AAD and then added to BMDMs expressing TLR3 YFP or TLR7 YFP . After incubation for 30 min at 37°C, the BMDMs were washed, and then the images were acquired as described under "Experimental Procedures." TLR3 YFP and TLR7 YFP are shown in green (pseudocolor), and apoptotic T cells are shown in red. The weakly stained nuclei with 7-AAD may represent apoptotic cells that were taken up by the macrophages earlier. Higher magnification panels to the right show the red, green, and merged channels separately for the boxed region.
sialyltransferase (23) and N-acetylglucosaminyltransferase I (24), are targeted to an intracellular compartment, such as the Golgi apparatus, by 17-25-amino acid sequences of their transmembrane domains by unknown mechanisms. It has also been reported that, in yeast, type II or type III transmembrane proteins, such as Sec12p or Sec71p, are retained in the ER by a Golgi protein, Rer1p (25,26). Sec12p and Sec71p contain a signal sequence for this targeting in their transmembrane domain. The polar amino acids in this targeting seem to be critical for recognition by Rer1p (26). Rer1p is a Golgi protein and is well conserved from yeast to mammals (27). It physically interacts with the transmembrane domain of Sec12p and promotes the retrieval of Sec12p from the Golgi to the ER (25,28). Thus, Rer1p works as a retrieval receptor recognizing the transmembrane domain of ER proteins in the Golgi. We found that Region 2 (Ser 844 -Val 850 ) of the transmembrane domain of TLR7 is the most important region for targeting TLR7 to an intracellular compartment. This region also contains four serine residues. One or more of these serine residues might be recognized by a Golgi protein, such as Rer1p. Molecular mechanisms of how signal sequences in transmembrane domains are recognized in the lipid bilayer, however, are still largely unknown, and further biochemical characterization is needed. TLR7, TLR8, and TLR9 are highly related in primary sequence and therefore constitute a TLR9 subfamily of Toll-like receptors (29). It was recently reported that TLR9 is localized to the ER (8). In addition, our previous results suggest that TLR8 is also mainly but not completely localized in an intracellular compartment (4). Since TLR8 and TLR9 have transmembrane domains that are similar to that of TLR7, they might also be targeted to an intracellular compartment such as the ER by their transmembrane domains.
When macrophages expressing YFP-tagged TLR3 or TLR7 were exposed to apoptotic cells, there was preferential localization of TLR3 and TLR7 adjacent to the phagocytosed apoptotic cells. During a virus infection, phagocytes take up apoptotic infected cells, and the action of lysosomal hydrolyases on the apoptotic cells presumably causes release of virus-derived RNAs, which are ligands for TLR3 and TLR7. Therefore, cell biological mechanisms for delivering TLR3 and TLR7 to the membranes of phagosomes containing apoptotic cells would likely be important for the function of these innate immune receptors. Our imaging studies are consistent with this hypothesis, as are findings that inhibitors of endosome and phagosome maturation, such as chloroquine and brefeldin A, block cellular responses to known ligands of TLR3 and TLR7 (5,7). Although the mechanism by which these inhibitors block the signaling initiated by these intracellular TLRs is unknown, they might in some way block the fusion of intracellular vesicles containing these TLRs with endosomes or phagosomes containing the added ligands.
The localization of different TLRs seems to be optimized for the recognition of their ligands. TLRs whose ligands are likely to be present outside host cells, such as those recognizing bacterial cell wall components, are localized on the cell surface, whereas TLRs whose ligands are released inside host cells, such as those recognizing nucleic acids, are localized intracellularly. Here we demonstrate that the localization of intracellular TLRs may be differentially controlled by distinct regulatory elements found either in the linker region between the transmembrane domain and the TIR domain or in the transmembrane region itself. Those elements lead TLRs to functionally useful positions in cells, where TLRs may access their ligands and induce responses for host defense.